In this proposal we describe a novel label-free assay that can be used to rapidly screen protein-small molecule interactions that are not easily screened by any other method. The label-free assay is based upon an optical biosensor technology called a "photonic crystal" structure that is inexpensively manufactured from continuous sheets of plastic film and incorporated into standard 96- and 384-well plates. By eliminating the need for a label, the assay is less susceptible to errors and artifacts caused by conformational change or blocking of active binding epitopes. Once fully developed, it is envisioned that the technology will be used in the context of a primary screen of a chemical library and/or as a secondary screen for measuring dose- response characteristics of a protein-small molecule combination. The proposed project develops a new type of label-free assay, called the "Spot Protocol," in which the volume of target protein required is <0.1 mu l/well, and error sources due to bulk refractive index variability and nonspecific binding are automatically eliminated. The new assay utilizes an image-based method to enable high sensitivity detection of small- molecule binding signals, but incorporates a novel image-processing algorithm to provide a simple numerical output that is a direct measurement of the binding interaction between protein and analyte. The successful completion of the proposed work would represent the first application of the photonic crystal assay technology to a high-throughput screening (HTS) application. By focusing the initial demonstrations on proteins that do not function as enzymes and are thus difficult to target through more standard HTS processes, the benefits of this technology towards the targeting of proteins traditionally viewed as "nondrugable" will be obvious. Thus, our goal is to develop this assay technology to the point where any researcher could use this label-free detection method to identify a small molecule ligand to a protein of interest, even if that protein in not an enzyme and thus has no conveniently-monitored activity-based readout. To make the community aware of the potential of such a method and thus facilitate its transfer into other laboratories, we have targeted proteins in the apoptotic pathway. By focusing on biochemical pathways that regulate apoptosis, the proposed project aims to make an impact on understanding the fundamental processes involved in programmed cell death, and on how misregulation of such processes are result in cancer and neurodegenerative disorders. The selection and identification of chemical compounds with the ability to selectively induce apoptosis of cancer cells, or to prevent apoptosis of healthy cells is one key to the development of new drug therapies for many diseases. [unreadable] [unreadable] [unreadable]